Stem cells are unspecialized cells that have two defining properties: the ability to differentiate into other cells and the ability to self-regenerate.
The ability to differentiate is the potential to develop into other cell types. A totipotent stem cell (e.g. fertilized egg) can develop into all cell types including the embryonic membranes. A pluripotent stem cell can develop into cells from all three germinal layers (e.g., cells from the inner cell mass). Other cells can be oligopotent, bipotent or unipotent depending on their ability to develop into few, two or one other cell type(s).
Self-regeneration is the ability of stem cells to divide and produce more stem cells. During early development, stem cell division is believed to be symmetrical i.e. each cell divides to gives rise to daughter cells, each with the same potential. Later in development, stem cells are believed to divide asymmetrically with one of the daughter cells produced being a stem cell and the other a more differentiated cell.
Stem cells are further classified according to their differentiation potential, roughly as follows:
Number of cellCell types resulting fromDifferentiation PotentialtypesExample of stem celldifferentiationTotipotentialAllZygote (fertilized egg),All cell typesblastomerePluripotentialAll except cells ofCultured human ESCells from all three germ layersthe embryoniccellsmembranesMultipotentialManyHematopoietic cellsskeletal muscle, cardiac muscle,liver cells, all blood cellsOligopotentialFewMyeloid precursor5 types of blood cells(Monocytes, macrophages,eosinophils, neutrophils,erythrocytes)Quadripotential4MesenchymalCartilage cells, fat cells, stromalprogenitor cellcells, bone-forming cellsTripotential3Glial-restricted2 types of astrocytes,precursoroligodendrocytesBipotential2Bipotential precursorB cells, macrophagesfrom murine fetal liverUnipotential1Mast cell precursorMast cellsNullipotentialNoneTerminallyNo cell divisiondifferentiated cell e.g.Red blood cell
As development proceeds, there is a loss of potential and a gain of specialization, a process called determination. For example, the cells of the germ layers are more specialized than the fertilized egg or the blastomere. The germ layer stem cells give rise to progenitor cells (also known as progenitors or precursor cells). For example, a cell in the endoderm gives rise to a primitive gut cell (progenitor), which can further divide to produce a liver cell (a terminally differentiated cell).
While there is consensus in the literature that a progenitor is a partially specialized type of stem cell, there are differences in how progenitor cell division is described. For instance, according to one source, when a stem cell divides at least one of the daughter cells it produces is also a stem cell; when a progenitor cell undergoes cell division it produces two specialized cells. A different source, however, explains that a progenitor cell undergoes asymmetrical cell division, while a stem cell undergoes symmetrical cell division.
The different kinds of human stem cells identified to date include: embryonic stem cells derived from embryos artificially produced in in-vitro fertility clinics, fetal stem cells derived from aborted fetuses, umbilical cord blood and placental blood stem cells, umbilical cord and placental tissue stem cells, bone marrow blood stem cells, peripheral blood stem cells, bone marrow mesenchymal stem cells, adult fat or adipose tissue-derived stem cells, cardiac muscle stem cells, skin epidermis stem cells, endothelial progenitor cells, brain and spinal cord derived neural stem cells, dental pulp stem cells and olfactory epithelium stem cells. In addition, human embryonic-like stem cells can be synthetically manufactured by inducing any adult terminally differentiated cell like a cheek skin fibroblast or even an adult stem cell into an embryonic-like stem cell.
Stem cells of the umbilical cord tissue are defined as mesenchymal stem cells or stromal stem cells with set characteristics. However, not all umbilical cord tissue cells have been tested for stem cell activity. Cord tissue mesenchymal stem cells have an ability to adhere to laboratory flask or dish surfaces and their morphology is fibroblast-like. They express specific surface markers like CD105, CD133, CD166, CD44, CD54, CD90, HLA-ABC, CD146, CD73, STRO-1 and are capable of differentiating into chondrocytes, adipocytes and osteocytes. Cord tissue mesenchymal stem cells are expected to engraft better than bone marrow mesenchymal stem cells or blood stem cells because unlike bone marrow mesenchymal stem cells and cord blood stem cells, they do not express a mature set of major histocompatibility antigens.
However, there are also inconsistencies in how stem cells are described and/or identified, making the field a challenging one to understand, and the literature is replete with inconsistencies.
The ability of stem cells to self-renew and give rise to subsequent generations with variable degrees of differentiation capacity offers significant potential to replace diseased and damaged areas in the body, with minimal risk of rejection and side effects. Many medical researchers believe that stem cell treatments have the potential to change the face of human disease and human tissue degeneration. Although unidentified back then, stem cells were used in tribal medicine thousands of years ago when tribe medical leaders looked for “young” blood to treat sick tribe members that would be regarded today as consanguineous people.
A number of stem cell therapies already exist, but most are at experimental stages or costly, with the notable exception of bone marrow and cord blood transplantations. Medical researchers anticipate that adult and embryonic stem cells will soon be able to treat cancer, Type 1 diabetes mellitus, Parkinson's disease, Huntington's disease, Celiac Disease, cardiac failure, muscle damage and neurological disorders, and many others. Nevertheless, before stem cell therapeutics can be applied in the clinical setting, more research is necessary to understand stem cell behavior after processing and upon transplantation as well as the mechanisms of stem cell interaction with the diseased/injured microenvironment.
Stem cells of different differentiation potentials are also defined by their source, age and method of preparation. For example, young cells are better in quality than old cells. Alternatively, cord blood is a better source of blood stem cells than bone marrow. On the other hand, time in culture, presence or lack of other cells in culture, xenogeneic or synthetic supplements and reagents alter biological properties of cells and stem cells including gain or loss of functions or specific properties like proliferation and differentiation potential.
In addition, for stem cells to be used in regenerative medicine one must consider two things, safety and efficacy. The method of preparation of cells and tissues for transplantations is very important because manipulating cells and tissue and introducing them to new agents, reagents and environments may turn these cells harmful or inefficient when transplanted in any individual, self or not. Further, current culture methods change stem cells in ways that can reduce or eliminate their efficacy and compromise their safety. Indeed, cord blood transplantation professionals complained about quality of cord blood units they receive from public and private banks and expect to transplant into patients as cord blood processing was not standardized and regulated by the US FDA. It was not until 2011 that cord blood became a licensed biological product. Except bone marrow and cord blood, all other cellular products remain in experimental stage and there is currently no FDA processing standard in place.
Methods to collect and preserve all types of stem cells fall into two basic categories—unmanipulated (or at least minimally manipulated) and manipulated methods.
The minimal manipulation method of collecting and freezing is mainly used for bone marrow and cord blood. The first minimal manipulation involved the collection of blood and direct infusion into a patient. Alternatively, aspirates of blood or marrow were mixed with blood anticoagulant and layered on specific density solutions like Ficoll-Hypaque to allow the density dependent separation of blood cells. The separation is accelerated by centrifugation. The middle “buffy coat” layer containing the blood stem cells is gently aspirated, leaving behind the top layer plasma and the bottom layer containing red blood cells. At this stage, the buffy coat is mixed with a cryoprotectant, like dimethyl sulfoxide (DMSO) or glycerol, to a final concentration of 10% and immediately slow frozen to −120° C. before immersing it in the liquid or gas phase of a liquid nitrogen storage tank or Dewar.
A new version of this method involves replacing the density solution with a set of processing bags connected through tubing and placed in a special device such as the AXP or SEPAX which is spun in a centrifuge leaving three final products, a red blood cell reduced concentration of mononuclear cells (buffy coat) containing blood and other stem cells, plasma, and red blood cells.
In other minimal manipulation methods, tissues are excised from one area of the body and retransplanted back in another of the same type. For example, skin from the leg area transplanted in an injured or burnt skin in the arm or face. Another example is scraped or shaved bone from the pelvis transplanted in a fractured or missing bone in the jaw.
The manipulated methods involve manipulating the cells longer than one hour and/or mixing the cells with agents other than water, phosphate buffer solution, cryoprotectant and Ficoll-Hypaque. Typically, in these methods, mechanical sectioning and/or enzymatic digestion of tissue to separate cells is used. Cells may be sorted, transfected for gene therapy and cultured in serum free media or media containing animal or genetically non-identical human sera, or genetically non-identical platelet lysates. Growth factors like epidermal growth factor and hormones like insulin may also be added to stimulate growth and proliferation of cultured cells. Furthermore, cells may be cultured in a two-dimensional or three-dimensional matrix where they may be guided to grow into a specific form. Alternatively, a piece of extracted tissue may be decellularized to create a matrix in which autologous or allogeneic cells may be infused. The matrix containing the necessary cells can be transplanted back into a patient to regrow and heal a degenerated tissue. All of these methods have a higher risk of negatively impacting stem cell safety and efficacy.
What is needed in the art are better methods isolating, culturing and preserving stems cells, that provides a more reliable, reproducible, safer and efficacious product.